Project group leader Kay Oliver SchinkPhosphoinositide control of early endocytic trafficking
Macropinocytosis is an endocytosis mechanism that leads to the formation of large vesicle filled with extracellular fluids and soluble macromolecules. Macropinocytosis is especially important in the immune system. Immune cells like dendritic cells and macrophages use this bulk uptake mechanism to sample the environment for foreign antigens to present them on their surface by MHC complexes. It is also exploited by many pathogens, which subvert this mechanism for cellular uptake and invasion. Moreover, cancer cells use macropinosomes as amino acids supply route to fuel their and amino-acid laden macropinosomes are major signaling hubs for mTORC1 signaling.
Formation of macropinosomes is a stochastic process that happens in areas of the cell that exhibit high levels of membrane reshaping. Rho GTPases trigger actin reorganization and formation of membrane ruffles, which can collapse, fuse and pinch off by yet unknown mechanisms to form large vesicles. These vesicles are then channeled into the endocytic pathway by recruitment of Rab5 and other endosomal proteins. During the transition from membrane ruffles to vesicles, the phosphoinositide composition of the ruffling plasma membrane changes - a phosphatase cascade metabolizes PtdIns(3,4,5)P3 by way of PtdIns(3,4)P2 and PtdIns(3)P to PtdIns. It appears that especially PtdIns(3)P, generated by dephosphorylation of PtdIns(3,4)P2, plays a critical role in the final step of macropinosome formation. This pool of PtdIns3P is clearly distinct from the Vps34-generated pool found at endosomes, and its biological function is still completely unexplored. After internalization, macropinosomes acquire Rab5 as well as a second, stable PtdIns3P pool by Vps34 kinase activity. Consequently, they enter the endolysosomal pathway, like other endocytic vesicles.
We aim to develop a deeper understanding how early steps of macropinocytosis work on a molecular level and how they are regulated. Of critical importance for this understanding will be to investigate the role of phosphoinositides and small GTPases of the Rho and Rab families and their regulators in this process. Macropinosomes offer an excellent model for phosphoinositide turnover on endocytic vesicles as they are large and easy to investigate in life cells, thereby allowing it to easily visualize different phosphoinositide pools.
We are currently investigating different steps of macropinosome formation and the cellular control mechanisms that drive successful or unsuccessful macropinocytosis
1: How do Rho GTPases and Phosphoinositides control macropinosome nucleation?
2: How do phosphatase-generated phosphoinositide pools and their effectors control early steps of macropinocytosis?
3: How do phosphoinositides and Rab GTPases control the identity of newly-formed macropinosomes?
We use a combination of molecular biology in combination with acute chemical and optogenetical perturbation approaches to study and disrupt phosphoinositide pools on newly-formed macropinosomes. We combine these methods with advanced life cell microscopy, single cell light sheet microscopy and super resolution microscopy.
Our research is supported by:
Kay Oliver Schink, Department of Molecular Cell Biology, Institute for Cancer Research
Oslo University Hospital,The Norwegian Radium Hospital,0379 Oslo, Norway
Phone +47 22 78 18 09